Effects Of Low Expression Of ABCG1 On Autophagy In Macrophages

BackgroundAtherosclerosis is a disease related to disorders of lipid metabolism.As a member of ATP-binding transporter superfamily,ABCG1(ATP-binding cassette G1)is a transmembrane transporter.It is mainly expressed in macrophages and participates in the reverse transport of intracellular cholesterol.ABCG1 mediate the transfer of the intracellular phospholipids and cholesterol(especially oxidized cholesterol)to the lipid receptor HDL to maintain lipid homeostasis.In the previous research of our team on the AB CG1 rs5713 7919 gene polymorphism,the risk and severity of coronary heart disease(CHD)patients with-367G>A mutation were significantly reduced,which is possibly relate to the acceleration of monocyte apoptosis caused by the low expression of ABCG1.We further explored in cells(in vitro)that ABCG1 knockdown can promote apoptosis,reduce oxidized cholesterol extracellular transfer and increase intracellular accumulation.Researchers currently suppose that the anti-atherosclerotic effect of ABCG1 with the low expression is related to the upregulation of pro-apoptotic genes induced by oxidized cholesterol accumulation and the compensatory mechanism of lipid metabolism.Therefore,this research is a continuation of previous research.In the present research,we use the THP-1 cell line to construct a cell line with low expression of ABCG1 through lentiviral infection.Thus,the aim of the research is to explore the low expression of ABCG1 effect on macrophage autophagy functions,so as to further explain the role of ABCG1 in atherosclerosis.ObjectiveThis study was to explore the effect of ARCG1 with low expression on autophagy function in THP-1 derived macrophages.Methods1.To construct the ABCG1 low expression cell line by using THP-1 cell line.Target cells were infected with lentiviral vectors,and stable transgenic strains were selected by puromycin.Eventually,the interference effects were verified in mRNA and protein levels by using Realtime-PCR and Western blot,separately.2.Western blot was used to detect the expression levels of the macrophage autophagosome membrane protein LC3 and the degradation substrate p62.The dynamic observation of autophagy flow induced by starvation.The immunofluorescence detect the fluorescence intensity of LC3 and p62.Western blot was used to evaluate the expression trend of LC3 and p62 and the expression differences of autophagy-related proteins(like Beclinl,ATG5,ATG7)under the induce of rapamycin.Results1.The results showed that ABCG1 decreased significantly in mRNA and protein levels in THP-1 Cell Lines,separately.2.In THP-1 derived macrophages with low ABCG1 expression,autophagy induced by starving.The expression levels of autophagosome membrane protein LC3 and autophagy degradation substrate p62 were significantly higher than negative control group(P<0.05).And the expression of LC3 and p62 increased further with the continuous extension of autophagy induced by time and reached a peak expression at 4 hours(P<0.05).Under the intervention of rapamycin,the expression level of LC3 in the knockdown group was still significant.However,the expression level of p62 in the knockdown group was significantly lower than negative control group(P<0.05).The expression of autophagy-related protein Beclinl was significantly increased in the knockdown group(P<0.01).While the expression of ATG5 and ATG7 decreased significantly(P<0.001).On the contrary,the expression of autophagy-related protein Beclinl in the knockdown group was significantly lower than negative control group(P<0.05)under rapamycin intervention.Conclusions1.THP-1 cell line with stable and low expression of ABCG1 was constructed.2.Autophagy function of THP-1-derived macrophages was blocked due to the low expression of ABCG1.This process did not affect the activation of autophagy and the formation of early autophagosomes.This process inhibited the specific degradation of late autophagosomes and was an mTOR dependent way.The mTOR-Beclinl pathway might be the target that affects the autophagy function of macrophages with low ABCG1 expression.